CN116253798B - Neutralizing monoclonal antibody for S1 protein conformational epitope of porcine delta coronavirus - Google Patents

Abstract

The invention belongs to the field of immunology and relates to a neutralizing monoclonal antibody directed against the conformational epitope of the S1 protein of porcine delta coronavirus, a hybridoma cell strain secreting the monoclonal antibody and its application. The heavy chain variable region of the neutralizing monoclonal antibody includes CDR1, CDR2 and CDR3 with amino acid sequences such as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; the light chain variable region includes the amino acid sequence CDR1, CDR2 and CDR3 as shown in SEQ ID NO:4, RAS, SEQ ID NO:5. The deposit number of the hybridoma cell line secreting the neutralizing monoclonal antibody is CCTCC NO: C2022248. The monoclonal antibody of the present invention has the clinical effect of treating and preventing pigs infected by porcine delta coronavirus, and can also be used to prepare porcine delta coronavirus detection reagents or kits.

Description

A neutralizing monoclonal antibody targeting the conformational epitope of porcine delta coronavirus S1 protein

Technical field

The invention belongs to the field of immunology, and specifically relates to a neutralizing monoclonal antibody directed against the conformational epitope of the S1 protein of porcine delta coronavirus, a hybridoma cell strain secreting the monoclonal antibody and its application.

Background technique

Porcine deltacoronavirus (PDCoV) is a newly discovered enteric coronavirus, a member of the genus Deltacoronavirus of the family Coronaviridae, which mainly causes vomiting, diarrhea, dehydration and death in newborn piglets. The clinical symptoms are similar to porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) and porcine transmissible gastroenteritis (Porcine transmissible gastroenteritis, TGE). So far, the occurrence and epidemic of PDCoV have been reported in more than 10 countries or regions around the world, posing a huge threat to the pig industry. PDCoV also has the potential to spread across species and can infect chickens, turkeys, cattle, mice and even Human beings, it has important public health significance and basic research value.

PDCoV is spherical with a diameter of 60nm to 180nm. Virus particles have an envelope, and the surface of the envelope has rod-shaped fibers formed by Spike(S) protein. The nucleocapsid has helical symmetry, is formed by a protein capsid wrapping a single-stranded RNA genome, and is located in the center of the virion (Ma Y M, Zhang Y, Liang X Y, et al. 2015. Origin, evolution, and virulence of porcine deltacoronaviruses in the United States .mBio,6(2):e00064.). Viruses contain four main structural proteins: spike protein S, small membrane protein E, membrane protein M and capsid protein N. The S protein, E protein and M protein together with the lipid bilayer constitute the virus envelope. The N protein makes up the capsid of the virus. The full length of the PDCoV genome is approximately 25.4 kb, with a cap structure and a non-coding region (5'UTR) at the 5' end, and a poly(A) tail and 3'UTR at the 3' end. The genome contains at least 9 open reading frames (ORFs), the order from 5' end to 3' end is ORF1a, ORF1b, S, E, M, NS6, N, NS7, NS7a, encoding a total of 15 mature Non-structural proteins (nsp2 ~ nsp16), 4 structural proteins (fibrillar protein S, small membrane protein E, membrane protein M and nucleocapsid protein N) and 3 accessory proteins (NS6, NS7 and NS7a). The gene encoding the accessory protein NS6 is located between the M gene and the N gene, the NS7 gene is located inside the N gene, and the NS7a gene is located at the C terminus of the NS7 gene (Fang P encoded by porcine deltacoronavirus.J Gen Virol,98(2):173-178.;Woo P C,Lau S K,Lam C S,et al.2012.Discovery of seven novel Mammalian and aviancoronaviruses in the genus deltacoronavirus supports bat coronaviruses as thegene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus. J Virol, 86(7):3995-4008.). The S protein of PDCoV is a type I membrane protein. It exists in the form of a trimer on the surface of the virus envelope and forms the fiber of the virus. Its main function is to recognize receptors, mediate the entry of the virus into host cells, and influence the host range and organization of the virus. The tropism plays a decisive role and is also the main protein that induces the production of neutralizing antibodies.

There are currently no commercial vaccines, diagnostic reagents, or therapeutic drugs for PDCoV, which poses a huge challenge to the effective prevention and control of the disease. Monoclonal antibodies with neutralizing activity can not only specifically recognize PDCoV, but also bind to specific neutralizing epitopes to prevent the interaction between the virus and host cells, thereby blocking infection and protecting the host. The preparation of monoclonal antibodies with PDCoV neutralizing activity can lay the foundation for the study of PDCoV neutralizing epitopes and the clinical prevention and treatment of PDCoV.

Contents of the invention

The purpose of the present invention is to provide a hybridoma cell strain that can stably secrete PDCoV-neutralizing monoclonal antibodies. The monoclonal antibodies effectively protect cells from being infected by PDCoV by neutralizing PDCoV, and further speculate that The monoclonal antibody has clinical effects in treating and preventing PDCoV-infected pigs.

In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

The present invention provides a hybridoma cell strain D-2H10 that can stably secrete PDCoV neutralizing monoclonal antibodies. The hybridoma cell strain has a deposit number of CCTCC NO: C2022248 and has good genetic stability.

The invention provides a PDCoV neutralizing monoclonal antibody D-2H10, which is secreted from the hybridoma cell strain D-2H10 with the deposit number CCTCC NO: C2022248. The heavy chain variable region of the PDCoV neutralizing monoclonal antibody D-2H10 includes CDR1 with the amino acid sequence GYTFTKYA (as shown in SEQ ID NO: 1), CDR2 with the amino acid sequence INPNNGGT (as shown in SEQ ID NO: 2) and The CDR3 with the amino acid sequence AGRMWFAF (as shown in SEQ ID NO:3); the light chain variable region of the PDCoV neutralizing monoclonal antibody D-2H10 includes CDR1 with the amino acid sequence ESVSFAGSIL (as shown in SEQ ID NO:4), The CDR2 whose amino acid sequence is RAS and the CDR3 whose amino acid sequence is MQSMEDPYT (as shown in SEQ ID NO: 5). The heavy chain variable region of PDCoV neutralizing monoclonal antibody D-2H10 is EVQLQQSGPELVKPGASVKISCKTSGYTFTKYAMHWVKQSHGKSLEWIGGINNPNNGGTT YNQKFKDKATLTVDKSSSTAYMELRSLTSEDSAVYYCAGRMWFAF (as shown in SEQID NO:6), and the light chain variable region is DIVLTQSPASLAVSLGQRATISCQASESVSFAGSIL MHWYQQKPGQPP KLLIYRASNLESGVPARFSGSGSESDFTLTIDPVEDDDAAMYVCMQSMEDPYT (as shown in SEQ ID NO: 7).

The present invention confirms through indirect immunofluorescence assay (Immunofluorescence Assay, IFA) that the monoclonal antibody can react specifically with PDCoV-infected LLC-PK1 cells.

The neutralizing titer of the purified monoclonal antibody D-2H10 of the present invention against PDCoV is 1:141. At this time, the concentration of the monoclonal antibody D-2H10 is 0.71 μg/mL; in the plaque reduction experiment, after purification The monoclonal antibody can better neutralize PDCoV and reduce the production of viral plaques.

Therefore, the monoclonal antibody D-2H10 or hybridoma cell line D-2H10 of the present invention can be used to prepare drugs for preventing or treating PDCoV infection, or to prepare swine delta coronavirus detection reagents or kits, or to prepare swine delta coronavirus detection reagents or kits, or for use in pigs. Scientific research on delta coronavirus. The single-chain antibody or antigen-binding fragment obtained by modifying the monoclonal antibody D-2H10 can also be used to prepare drugs to prevent or treat PDCoV infection, or to prepare porcine delta coronavirus detection reagents or kits; or for porcine delta coronavirus Scientific research on coronavirus.

Indirect immunofluorescence experiments and Western-blot detection confirmed that the antigenic epitope recognized by the monoclonal antibody D-2H10 of the present invention is the conformational epitope of the PDCoV-S1 protein.

The present invention also provides gene sequences encoding the heavy chain variable region and light chain variable region of the PDCoV neutralizing monoclonal antibody D-2H10, encoding the heavy chain variable region and light chain variable region of the monoclonal antibody D-2H10. The gene sequence of the chain variable region was obtained by amplifying the degenerate primers of the murine antibody variable region using the cDNA of the hybridoma cell line D-2H10 with the deposit number CCTCC NO: C2022248 as a template. The gene sequence encoding the heavy chain variable region of monoclonal antibody D-2H10 is 5'-GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGA AGATATCCTGCAAGACTTCTGGATACACATTCACTAAATACGCCATGCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGGTATTAATCCTAACAATGGTGGTACTACTTACAACCAGAAGTTCAAGGACAAGGCCACATTGACT GTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGATTCTGCAGTCTATTACTGTGCAGGGAGAATGTGGTTTGCTTTC-3', as shown in SEQ ID NO:8, a gene encoding the light chain variable region of monoclonal antibody D-2H10 The sequence is 5'-GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCAG TGTCTCTAGGACAGAGGGCCACCATTTCCTGCCAAGCCAGCGAAAGTGTCAGTTTTGCTGGTTCAATTTTAATGCACTGGTACCAACAGAAACCAGGACAGCCACCGAAACTCCTCATCTATCGTGCATCCAACCTAGAATCTGGAGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGAGTCAGACTTCACTCTCACCATCG ATCCTGTGGAGGATGATGATGCGGCAATGTATGTCTGTATGCAAAGTATGGAAGATCCGTACACG-3', as shown in SEQ ID NO:9.

The invention has the following beneficial effects:

The hybridoma cell line D-2H10 of the present invention can stably secrete the PDCoV neutralizing monoclonal antibody D-2H10. In addition, the monoclonal antibody D-2H10 of the present invention effectively protects cells from being infected by PDCoV by neutralizing PDCoV. Therefore, the monoclonal antibody D-2H10 has the clinical effect of treating and preventing PDCoV-infected pigs.

Description of the drawings

Figure 1 is a technical roadmap of the present invention.

Figure 2 shows the SDS-PAGE results of purified virus. Explanation of reference signs: M: Protein Marker; 1: sample at the stratified location of 30% to 45% sucrose; 2: sample at the stratified location of 45% to 60% sucrose.

Figure 3 shows the Western-blot results of the purified virus. Explanation of reference signs: M: Protein Marker; 1: sample at the stratified location of 30% to 45% sucrose; 2: sample at the stratified location of 45% to 60% sucrose.

Figure 4 shows IFA verification that monoclonal antibody D-2H10 specifically recognizes PDCoV. Explanation of reference numbers: A: LLC-PK1 cells infected with PDCoV; B: LLC-PK1 cells not infected with virus control.

Figure 5 shows the plaque reduction assay to detect the neutralizing ability of monoclonal antibody D-2H10 against PDCoV. Explanation of reference symbols: A to E: monoclonal antibody D-2H10 experimental group (A: 4 μg/mL monoclonal antibody D-2H10, B: 2 μg/mL monoclonal antibody D-2H10, C: 1 μg/mL monoclonal antibody D-2H10, D: 0.5 μg/mL monoclonal antibody D-2H10, E: 0.25 μg/mL monoclonal antibody D-2H10); F: monoclonal antibody control; G: virus control.

Figure 6 shows the PDCoV epitope recognized by monoclonal antibody D-2H10 identified by IFA. Explanation of reference symbols: A: PDCoVS; B: PDCoV S1; C: PDCoV S1-NTD; D: PDCoV S1-CTD.

Figure 7 shows Western-blot identification of the PDCoV epitope recognized by monoclonal antibody D-2H10. Explanation of reference symbols: M: Protein Marker; 1: purified PDCoV; 2: PDCoV S.

deposit information

Hybridoma cell line D-2H10:

Storage time: August 18, 2022;

Name of preservation institution: China Typical Culture Collection Center;

Deposit number: CCTCC NO: C2022248;

Address of the depositary institution: Wuhan University, Wuhan, China;

Classification and naming: hybridoma cell line D-2H10.

Detailed ways

The present invention will be described in more detail below through specific embodiments to facilitate understanding of the technical solution of the present invention, but is not intended to limit the scope of protection of the present invention.

The PDCoV virus liquid of the present invention is obtained by infecting LLC-PK1 cells with PDCoV, and the antigen used for animal immunity is the virus particles obtained by purifying the expanded cultured PDCoV virus liquid through conventional sucrose gradient centrifugation in this field.

The hybridoma cell line D-2H10 of the present invention, which can stably secrete PDCoV neutralizing monoclonal antibodies, has a deposit number of CCTCC NO: C2022248, and is obtained by fusion of mouse splenocytes immunized with PDCoV virus fluid and myeloma cells SP2/0. .

The PDCoV neutralizing monoclonal antibody D-2H10 of the present invention is secreted from the hybridoma cell line D-2H10. Antibody purification adopts conventional ascites-type monoclonal antibody preparation and purification methods in the field. The ELISA titer of the ascites-type antibody of the monoclonal antibody D-2H10 is 1:100×2 ⁷ .

The monoclonal antibody D-2H10 of the present invention can react specifically with PDCoV-infected LLC-PK1 cells, but does not react with LLC-PK1 cells not infected with PDCoV, indicating that the monoclonal antibody D-2H10 can react specifically with PDCoV. reaction.

In the present invention, the neutralization experimental method is preferably the "fixed virus-dilute serum" method, and LLC-PK1 cells are preferred. The results show that: calculated according to the Reed-Muench method, the neutralizing titer of the purified monoclonal antibody D-2H10 is 1 :141, at this time the concentration of monoclonal antibody D-2H10 is 0.71μg/mL. In the plaque reduction experiment, the results showed that: compared with the PDCoV virus control group and the PRRSV N protein monoclonal antibody + PDCoV control group, the number of viral plaques in the monoclonal antibody D-2H10 experimental group was significantly reduced, and the number of viral plaques was reduced. The quantity is positively correlated with the dosage of monoclonal antibody D-2H10. In the present invention, the monoclonal antibody D-2H10 effectively protects cells from being infected by PDCoV by neutralizing PDCoV. It is further speculated that the monoclonal antibody D-2H10 may have clinical effects in treating and preventing PDCoV-infected pigs.

In the antigenic epitope identification experiment of the monoclonal antibody D-2H10 of the present invention, the eukaryotic expression plasmids involved in PDCoV S, PDCoV S1, PDCoV S1-NTD, and PDCoV S1-CTD were all constructed and stored in the inventor's laboratory. The DNA sequences encoding PDCoV S, PDCoV S1, PDCoV S1-NTD, and PDCoV S1-CTD are shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 14 respectively; the amino acids of PDCoV S1 The sequence is, as shown in SEQ ID NO:12. In the present invention, HEK293T cells are transfected with recombinant eukaryotic expression plasmids PDCoV S, PDCoV S1, PDCoV S1-NTD, and PDCoV S1-CTD, and monoclonal antibody D-2H10 is used as the primary antibody for IFA detection. The results show: The monoclonal antibody can react specifically with the PDCoV S protein and PDCoV S1 protein expressed in the transfected cells, but has no specific reaction with the PDCoV S1-NTD and PDCoV S1-CTD proteins expressed in the transfected cells, indicating that the monoclonal antibody D -2H10 can specifically recognize the PDCoV S1 protein, and it is speculated that the epitope recognized by the monoclonal antibody D-2H10 may be the conformational epitope of the PDCoV S1 protein.

Purified PDCoV and the overexpressed PDCoV S protein were used for SDS-PAGE electrophoresis, and monoclonal antibody D-2H10 was used as the primary antibody for Western-blot detection. The results showed that the monoclonal antibody D-2H10 and purified PDCoV and There was no specific response to the eukaryotic expressed PDCoV S protein, indicating that the epitope specifically recognized by the monoclonal antibody was the conformational epitope of the PDCoV S1 protein.

The heavy chain variable region and light chain variable region gene sequences of the PDCoV neutralizing monoclonal antibody D-2H10 provided by the invention are based on the total RNA of the hybridoma cell line D-2H10 reversed into cDNA as a template , and then obtained by PCR amplification and sequencing using degenerate primers for the heavy chain and light chain of the murine antibody variable region. The technical roadmap of the present invention is shown in Figure 1.

The antigens, hybridoma cells, and monoclonal antibodies in the present invention will be described in detail below with reference to specific examples.

Example 1 Preparation of Antigen

1. Massive amplification of PDCoV

LLC-PK1 cells were transferred to a ^175cm2 cell culture flask, and the culture medium was MEM containing 10% FBS, and cultured at 37°C and 5% _CO2 until the cells grew into a monolayer. Rinse the cell monolayer twice with FBS-free MEM medium containing 7.5 μg/mL trypsin, add FBS-free MEM medium containing 7.5 μg/mL trypsin, and incubate PDCoV at an MOI of 0.1 to 0.5. Inoculate and culture at 37°C, 5% _CO2 . When the cell lesions reach 80%, freeze and thaw 2 to 3 times, centrifuge at 4000r/min for 15min to remove cell debris, and harvest the supernatant to obtain PDCoV virus liquid.

2. Purification of PDCoV whole virus particles

Centrifuge the PDCoV virus liquid at 8000 r/min for 45 min, take the supernatant, and filter it with a 0.8 μm filter to remove cell debris. Add NaCl with a final concentration of 0.5 M and polyethylene glycol 6000 (PEG 6000) with a final concentration of 5% to the filtered virus liquid. Mix thoroughly and precipitate at 4°C for 24 hours. Centrifuge at 12000 r/min for 1 hour and discard. Then add appropriate amount of PBS to the pellet and resuspend at 4°C overnight. Add the virus resuspended in PBS to the upper part of the ultra-high-speed centrifuge tube pre-added with 30%, 45% and 60% sucrose solutions, centrifuge at 36000r/min for 3 hours, and aspirate the 30% to 45% sucrose solution layer. A ring-shaped layer of virus. Place the sample in a new ultra-high-speed centrifuge tube, fill the centrifuge tube with PBS, mix gently by pipetting, and centrifuge at 35,000 r/min for 2 hours. Discard the supernatant and add a small amount of PBS to the pellet to resuspend. The resuspended virus liquid was subjected to SDS-PAGE detection.

The results showed that obvious bands appeared at about 170KDa, 40KDa, and 25KDa, which were consistent with the sizes of PDCoV S protein, N protein, and M protein respectively; as shown in Figure 2.

Further, the monoclonal antibody of the PDCoV N protein prepared and stored in our laboratory was used as the primary antibody to detect the purified virus through Western-blot. The results showed that a single specific band appeared at about 40KDa in size, as shown in Figure 3 Show. The above results indicate that viruses with higher purity were obtained. The purified virus liquid was aliquoted and stored at -80°C for later use.

Example 2 Preparation of monoclonal antibodies

1. Establishment of hybridoma cell line D-2H10

The purified PDCoV was mixed with an equal volume of rapid immune adjuvant (purchased from Boaolong Immunology Technology Co., Ltd.) and injected into 6- to 8-week-old female BALB/c mice through the hind leg muscles. After three times of immunization, PDCoV- The ELISA method (indirect ELISA established with purified PDCoV as the antigen) detects PDCoV antibodies in mouse serum. When the antibody titer reaches 1:12800, purified PDCoV is used for booster immunization. 3 to 5 days after the boosted immunization, the mice were sacrificed by bleeding from the orbit. At the same time, the blood was collected and the serum was separated, which was regarded as positive serum. Spleen cells from immunized mice were aseptically fused with myeloma cells SP 2/0. After culturing in HAT medium and HT medium, the culture supernatant of the fused cells was detected by PDCoV-ELISA method. The positive wells were The cells were subjected to 3 rounds of subcloning and screening. After the positive cell lines were expanded and cultured, the culture supernatants were used for neutralization tests. Finally, a hybridoma cell line that could stably secrete PDCoV neutralizing monoclonal antibodies was obtained. It was named D-2H10.

The applicant has sent the hybridoma cells to the Chinese Type Culture Collection Center of Wuhan University for preservation on August 18, 2022, with the preservation number CCTCC NO: C2022248.

2. Large-scale preparation and purification of monoclonal antibody D-2H10

(1) Large-scale preparation of monoclonal antibody D-2H10: Select three 10-week-old healthy female BALB/c mice, intraperitoneally inject Freund's incomplete adjuvant, 500 μL/mouse, and 7 days later, intraperitoneally inject hybridoma cell line D- 2H10, 5×10 ⁵ ~ 1×10 ⁶ cells/cell. After 10 to 14 days, the ascites of the mice was collected according to the abdominal distension of the mice. Centrifuge the collected ascites at 4°C and 12,000 r/min for 10 minutes, and collect the clear yellow liquid in the middle to obtain ascites-type monoclonal antibodies.

(2) Titer detection of ascites-type monoclonal antibody D-2H10: Use the PDCoV whole virus particles prepared in the present invention to coat an enzyme-labeled plate, and dilute the ascites-type monoclonal antibody from a volume ratio of 1:100×2 times to 1 :100×2 ¹² as the primary antibody, and HRP-labeled goat anti-mouse IgG as the secondary antibody for indirect ELISA detection. The results show that the ELISA titer of the ascites-type antibody of the monoclonal antibody D-2H10 of the present invention is 1:100×2 ⁷ .

(3) Purification of ascites-type monoclonal antibody D-2H10: Purify the prepared ascites-type monoclonal antibody D-2H10 using a Protein G column (purchased from Fuyinder Technology Co., Ltd.) according to the instructions, and then purify the purified monoclonal antibody D-2H10. Antibody D-2H10 was used for SDS-PAGE detection.

The results showed two protein bands, light chain and heavy chain, indicating that a monoclonal antibody with high purity was obtained.

Example 3 IFA detection of the reaction between monoclonal antibody D-2H10 and PDCoV

LLC-PK1 cells were seeded in a 24-well plate. After the cells had grown to cover the monolayer, the cells were infected with PDCoV according to conventional methods. After the cells appeared lesions, the cell culture supernatant was discarded and 1 mL of tissue fixative (4) was added to each well. % paraformaldehyde (purchased from Biosharp Company), fixed for 15 minutes, added -20°C pre-cooled methanol, permeabilized for 10 minutes, washed three times with PBS, 5 minutes each time. Add PBS containing 5% BSA, block for 1 hour at 37°C, and wash three times with PBS for 5 minutes each time. Add purified monoclonal antibody D-2H10 (1000-fold dilution with PBS), 250 μL per well, and incubate at 37°C for 1 hour. Wash 3 times with PBS, 5 min each time. Add 250 μL of fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG (purchased from Beyotime Biotechnology Co., Ltd.) (2000-fold dilution with PBS) to each well, and incubate at 37°C for 1 hour, protected from light throughout. Add 250 μl DAPI (purchased from Beyotime Biotechnology Co., Ltd.) to each well (5000-fold dilution with PBS), and incubate at 37°C for 15 min. Aspirate away the fluorescent secondary antibody and DAPI diluent, wash 3 times with PBS, add 200 μl PBS to each well, observe and take pictures under an inverted fluorescence microscope.

The results show that the monoclonal antibody D-2H10 prepared in the present invention has a specific fluorescence reaction with PDCoV-infected LLC-PK1 cells, but has no fluorescence reaction with LLC-PK1 cells not exposed to the virus, as shown in Figure 4, illustrating the preparation of the present invention The monoclonal antibody D-2H10 specifically recognizes PDCoV in IFA.

Example 4 Detection of neutralizing activity of purified monoclonal antibody D-2H10 against PDCoV

1. Neutralization test (fixed virus-dilute serum method) to detect the neutralizing activity of monoclonal antibodies

PDCoV was diluted to 200TCID ₅₀ /0.1mL using serum-free MEM medium containing 7.5μg/mL trypsin, and the purified monoclonal antibody D-2H10 was diluted 2-fold starting from 100μg/mL to 0.048μg/mL. Mix monoclonal antibody D-2H10 at different dilutions with an equal volume of 200TCID ₅₀ /0.1mL virus liquid and place at 37°C for 1 hour. Wash the monolayer LLC-PK1 cells in a 96-well cell culture plate twice with serum-free MEM culture medium containing 7.5 μg/mL trypsin, and inoculate the antibody-virus mixture into the 96-well plate at each antibody dilution. Inoculate 4 wells, 100 μL per well, set 200TCID ₅₀ /0.1mL, 20TCID ₅₀ /0.1mL, 2TCID ₅₀ /0.1mL and 0.2TCID ₅₀ /0.1mL virus control, place it in a 37°C cell culture incubator containing 5% _CO2 After 1 hour, the cell monolayer was washed twice with serum-free MEM culture medium containing 7.5 μg/mL trypsin, and 100 μL of serum-free MEM culture medium containing 7.5 μg/mL trypsin was added to each well, and placed at 37°C with 5 In a % CO ₂ cell culture incubator, observe and record the cytopathic effects (CPE) every day until the cytopathic effects are stable (about 72 hours), and calculate the neutralizing titer of the antibody against PDCoV according to the Reed-Muench method.

Neutralization experiment results show that the neutralization titer of the purified monoclonal antibody D-2H10 is 1:141, and the concentration of the monoclonal antibody D-2H10 at this time is approximately 0.71 μg/mL.

2. Plaque reduction assay to detect the neutralizing activity of monoclonal antibodies

PDCoV was diluted to 200TCID ₅₀ /0.1mL in serum-free MEM medium containing 7.5μg/mL trypsin, and the purified monoclonal antibody D-2H10 was diluted 2-fold starting from 4μg/mL to 0.25μg/mL. Mix monoclonal antibody D-2H10 at different dilutions with an equal amount of 200TCID ₅₀ /0.1mL virus liquid and place at 37°C for 1 hour. Wash the LLC-PK1 cells that have grown into a monolayer in a 6-well cell culture plate twice with serum-free MEM culture medium containing 7.5 μg/mL trypsin, and inoculate the antibody-virus mixture into the 6-well plate, with 2 mL per well. Set up a monoclonal antibody control (PRRSV N protein monoclonal antibody (prepared and stored by the inventor's laboratory) + PDCoV) and a virus control (200TCID ₅₀ /0.1mL of PDCoV), and place them in a 37°C cell culture containing 5% _CO2 After 1 hour in the chamber, the cell monolayer was washed twice with serum-free MEM culture medium containing 7.5 μg/mL trypsin. Mix 2% low melting point agarose with an equal volume of phenol red-free 2×DMEM containing 15 μg/mL trypsin and add it to the cell plate. Place it at 4°C until the agarose solidifies. Incubate the cell culture plate at 37°C in the presence of 5 μg/mL trypsin. Culture in % CO ₂ cell incubator. After the plaque appears, fix it with 4% paraformaldehyde and leave it at room temperature for 15-30 minutes. Aspirate and discard the fixative, add crystal violet staining solution, leave it at room temperature for 1 to 2 hours, discard the agarose and staining solution, and rinse gently with running water. , count and count the virus plaques after drying.

The results showed that the results of the monoclonal antibody D-2H10 experimental group were significantly reduced compared with the PDCoV virus control group and the PRRSV N protein monoclonal antibody + PDCoV control group, and the number of reduced plaques was consistent with the monoclonal antibody D- The dosage of 2H10 is positively correlated. When the concentration of monoclonal antibody D-2H10 reaches 2 μg/mL, the appearance of plaques can be completely blocked, as shown in Figure 5, indicating that the monoclonal antibody D-2H10 prepared by the present invention can effectively protect cells. Protects against PDCoV infection, thereby reducing the formation of viral plaques.

Example 5 Identification of the specific recognition antigen epitope of monoclonal antibody D-2H10

1. IFA identifies the epitope recognized by monoclonal antibody D-2H10

The recombinant plasmids expressing PDCoV S and PDCoV S1 were transfected into HEK293T cells respectively. After 24 hours, the cell culture medium was aspirated and IFA identification was performed according to the method in Example 3.

The results show that the monoclonal antibody D-2H10 prepared in the present invention has a specific fluorescence reaction with cells transfected with PDCoV S or PDCoV S1 recombinant plasmid, indicating that the D-2H10 monoclonal antibody prepared in the present invention interacts with PDCoV S recombinant protein and PDCoV S1 recombinant proteins all have good specific reactions.

The recombinant plasmids expressing PDCoV S1-NTD and PDCoV S1-CTD were transfected into HEK293T cells respectively. After 24 hours, the cell culture medium was aspirated, and IFA identification was performed according to the method in Example 3.

The results showed that monoclonal antibody D-2H10 had no specific fluorescence reaction with the expressed PDCoV S1-NTD recombinant protein and PDCoV S1-CTD recombinant protein, as shown in Figure 6. This shows that the epitope recognized by monoclonal antibody D-2H10 may be the conformational epitope of PDCoVS1 protein.

2. Western-blot identification of the epitope recognized by monoclonal antibody D-2H10

The recombinant plasmid expressing PDCoV S protein was transfected into HEK293T cells. 24 hours after transfection, the recombinant protein sample was collected and subjected to SDS-PAGE electrophoresis with the PDCoV purified virus prepared in the present invention. After electrophoresis, transfer to PVDF (Polyvinylidene Fluoride) membrane using wet transfer method. Block with TBST containing 5% skim milk at room temperature for 2 h, and then wash 3 times with TBST buffer. Add an appropriate amount of monoclonal antibody D-2H10, incubate at room temperature for 2 hours, wash 3 times with TBST buffer, then add an appropriate amount of HRP-labeled goat anti-mouse IgG as a secondary antibody, incubate at room temperature for 1 hour, wash 3 times with TBST buffer, and then add an appropriate amount of Chromogenic solution for color development on a chemiluminescence imager.

The results show that the monoclonal antibody D-2H10 prepared in the present invention has no specific reaction with PDCoV and the expressed PDCoV S recombinant protein in Western-blot detection, as shown in Figure 7.

The above results show that the monoclonal antibody D-2H10 prepared in the present invention can be detected by IFA to have specific fluorescence reaction with the whole PDCoV virus, the eukaryotic expressed PDCoV S recombinant protein and the PDCoV S1 recombinant protein, but it does not react with the eukaryotic expressed PDCoV S1 truncated body (PDCoV S1-NTD, S1-CTD) showed no reaction; at the same time, Western-blot detection found that the monoclonal antibody D-2H10 prepared in the present invention had no specific reaction with the PDCoV whole virus and the eukaryotic expressed PDCoV S recombinant protein. . From this, it can be determined that the epitope recognized by monoclonal antibody D-2H10 is the conformational epitope of PDCoV S1 protein.

Example 6 Amplification and analysis of variable region gene of monoclonal antibody D-2H10

The total RNA of hybridoma cell D-2H10 was extracted and reversed to cDNA as a template. 11 pairs of degenerate primers were designed for amplification of the light chain variable region gene of monoclonal antibody D-2H10. 12 pairs of degenerate primers were designed for monoclonal antibody D-2H10 light chain variable region gene amplification. Amplification of the heavy chain variable region gene of cloned antibody D-2H10.

Table 1 Primer sequences for amplifying light chain variable regions

Light chain primer name Primer sequence (5’～3’) MKV1 ATGAAGATTGCCTGTTAGGCTGTTGGTGCTG MKV2 ATGGAGWCAGACACACTCCTGYTAYGGGTG MKV3 ATGAGTGTGCTCACTCAGGTCCTGGSGTTG MKV4 ATGAGGRCCTGCTCAGWTTYTTGGMWTCTTG MKV5 ATGGATTTWCAGGGTGCAGATTWTCAGCTTC MKV6 ATGAGGTKCYYTGYTSAYCTYCTCTGRGG MKV7 ATGGGCWTCAAAGATGGAGTCACAKWYYCWGG MKV8 ATGTGGGGAYCTKTTTYCMMTTTTTCAATG MKV9 ATGGTRTCCWCASCTCAGTTCCTTG MKV10 ATGTATATATGTTTGTTGTCTATTTCT MKV11 ATGGAAGCCCCAGCTCAGCTTCTCTTCC MKC ACTGGATGGTGGGAAGATGG

Table 2 Primer sequences for amplifying the heavy chain variable region

The results showed that there was one pair of primers (MKV2, MKC) that could amplify the light chain variable region gene, and there were two pairs of primers (MHV5/MHV7, MHCG1) that could amplify the heavy chain variable region gene. The obtained amplified fragments were connected to the T vector and sequenced to obtain one antibody light chain variable region gene sequence and two antibody heavy chain variable region gene sequences (one of which belongs to an ineffective rearrangement gene containing a stop codon) . The light chain variable region sequence is 303bp in size and has a complete antibody variable region structure. Positions 23 and 92 are the characteristic amino acids - cysteine, which has 96.91% homology with Musmus IGKV3-9*01F; heavy The chain variable region sequence is 312bp in size and has a complete antibody variable region structure. Positions 22 and 96 are the characteristic amino acids - cysteine, which has 95.49% homology with MusmusIGHV1-18*01F.

The heavy chain variable region of monoclonal antibody D-2H10 includes CDR1 with an amino acid sequence as shown in SEQ ID NO:1, CDR2 with an amino acid sequence as shown in SEQ ID NO:2, and CDR2 with an amino acid sequence as shown in SEQ ID NO:3. CDR3; the light chain variable region of the PDCoV neutralizing monoclonal antibody D-2H10 includes CDR1 with the amino acid sequence shown in SEQ ID NO:4, CDR2 with the amino acid sequence RAS and the amino acid sequence shown in SEQ ID NO:5 CDR3. The heavy chain variable region of the PDCoV neutralizing monoclonal antibody D-2H10 is shown in SEQ ID NO: 6, and the light chain variable region is shown in SEQ ID NO: 7.

The gene sequence encoding the heavy chain variable region of monoclonal antibody D-2H10 is shown in SEQ ID NO:8, and the gene sequence encoding the light chain variable region of monoclonal antibody D-2H10 is shown in SEQ ID NO:9.

The above-described embodiments are only preferred embodiments of the present invention and do not limit the scope of the present invention. Therefore, any equivalent changes or modifications based on the structures, features and principles described in the patent scope of the present invention are should be included in the patent scope of this invention.

Claims (9)

1. The neutralizing monoclonal antibody aiming at the conformational epitope of the S1 protein of the porcine delta coronavirus is characterized in that a heavy chain variable region of the neutralizing monoclonal antibody comprises a CDR1 with an amino acid sequence shown as SEQ ID NO. 1, a CDR2 with an amino acid sequence shown as SEQ ID NO. 2 and a CDR3 with an amino acid sequence shown as SEQ ID NO. 3; the light chain variable region of the neutralizing monoclonal antibody comprises a CDR1 with an amino acid sequence shown as SEQ ID NO. 4, a CDR2 with an amino acid sequence of RAS and a CDR3 with an amino acid sequence shown as SEQ ID NO. 5.

2. The neutralizing monoclonal antibody of claim 1 wherein the heavy chain variable region of the neutralizing monoclonal antibody is set forth in SEQ ID No. 6 and the light chain variable region is set forth in SEQ ID No. 7.

3. DNA encoding the neutralizing monoclonal antibody of claim 1 or 2.

4. The DNA according to claim 3, wherein the DNA encoding the heavy chain variable region of the neutralizing monoclonal antibody is shown in SEQ ID NO. 8 and the DNA encoding the light chain variable region of the neutralizing monoclonal antibody is shown in SEQ ID NO. 9.

5. A biological material comprising the DNA of claim 3 or 4, said biological material being an expression cassette, a transposon, a plasmid vector, a viral vector, an engineered bacterium or a host cell.

6. The single chain antibody or antigen binding fragment of claim 1 or 2 engineered from a neutralizing monoclonal antibody.

7. A medicament, detection reagent or kit comprising the neutralizing monoclonal antibody of claim 1 or 2 or the single chain antibody or antigen binding fragment of claim 6.

8. A hybridoma cell strain secreting a neutralizing monoclonal antibody against a conformational epitope of porcine delta coronavirus S1 protein, wherein the hybridoma cell strain has a preservation number of CCTCC NO: C2022248.

9. the neutralizing monoclonal antibody of claim 1 or 2 or the single chain antibody or antigen binding fragment of claim 6 for any one of the following uses:

a: for preparing a medicament for preventing or treating diseases related to infection of porcine delta coronavirus and infection thereof;

b: the method is used for preparing the detection reagent or the kit for the porcine delta coronavirus.